Neurons communicate with each other by transmitting chemical signals from cell to cell. In the key process of neuronal exocytosis, a voltage wave travels the length of the transmitting cell to open Ca2+ channels in the plasma membrane (PM) at the axon terminus. The abrupt influx of Ca2+ triggers exocytosis, the process by which secretory vesicles docked at the PM open a fusion pore and spill their neurotransmitter into the synaptic cleft. The neurotransmitter is taken up by receptors placed on the opposite side of the synapse in the PM of the receiving neuron's dendrite. This in turn stimulates a second voltage wave that travels the length of the receiving neuron. The same process of exocytosis enables endocrine cells to emit triggered chemical signals into the intercellular space or the bloodstream. The vesicle fusion machine is the complex of proteins that cause exocytosis. Key components include the SNARE (soluble NSF attachment receptor) proteins syntaxin (Syx), SNAP-25, and synaptobrevin (Syb), and the Ca2+ sensor synaptotagmin (Syt). Essentially nothing is known about the stoichiometry (number of working copies of each protein) or the overall architecture (spatial relationships among the proteins) of the fusion machine, either before or after the Ca2+ influx. The goal of this work is to use the new fluorescence technique of photoactivated localization microscopy (PALM) to count and to locate fusion machine proteins with ~5-nm accuracy in fixed PC-12 cells, a standard model for the study of exocytosis. PALM uses a long sequence of activation-location-photobleaching cycles to locate all labeled copies of a protein one by one. In each experiment, the photoactivatable fluorescent protein mEos will be genetically attached to a particular fusion protein (Syx, SNAP-25, Syb, or Syt). For each cell studied, the LDCVs (large, dense-core secretory vesicles) will also be located to ~5 nm by co-expression of the neuropeptide eGFP-ANF, which trafficks to LDCVs and exocytoses normally. Comparison of PALM images of resting cells vs depolarized cells may reveal structural changes induced by the Ca2+ influx. New structural information is critically important to progress in understanding the underlying molecular mechanism of triggered vesicle fusion. PALM imaging has the potential to reveal the structure of the fusion machine before and after Ca2+ influx at an entirely new level of detail. Existing hypotheses about rings of Syx/SNAP-25 centered beneath a docked vesicle and about binding relationships between Syt and Syx, among many others, will be tested directly. In the longer term, extensions to PC-12 cells differentiated by culture in neuron growth factor (NGF) and to hippocampal neurons are envisioned. The new methodology developed here should find widespread application in structural studies of other large protein complexes embedded in cellular membranes. PUBLIC HEALTH RELEVANCE: The goal of this work is to use a new high-resolution fluorescence microscopy technique to determine the number and arrangement in space of key proteins in the vesicle fusion machine. That machine is the complex of proteins that controls the release of neurotransmitters into the synapse, the key step in neuron-neuron communication. In the long run, a fundamental understanding of how neurons work at the molecular level should help elucidate the underlying causes of neurodegenerative diseases such as Parkinson's and Alzheimer's disease.